Chronic muscle diseases including Duchenne muscular dystrophy (DMD) and aging-related sarcopenia result in muscle weakness, loss of independence, and increased risk of death. In addition, traumatic muscle injury and loss due to accidents, surgery, and wartime injuries needs prolonged recovery. Skeletal muscle is a highly regenerative tissue in which satellite cells, a stem cell population for skeletal muscle, play essential roles in creating and repairing skeletal muscle. However, this potential ultimately fails with disease and aging. Autologous satellite cell transplantation is a potential approach to create and repair skeletal muscle fibers, but satellite cells are rare (a few % of all muscle nuclei) and often difficult to isolate. Patient-derived induced pluripotent stem cells (iPSCs) are the ideal cell source to obtain an unlimited number of myogenic cells that escape immune rejection after engraftment. However, efficient myogenic differentiation and the scale-up of myogenic differentiation remain elusive and must be developed further in order to generate effective cellular therapies. A key to the generation of human myogenic cells and skeletal muscle in a host animal is the selective knockout of genes in the blastocyst that are critical for organ development. Therefore, in this proposal, (1) we will determine to which extent mouse iPSC-derived limb skeletal muscle will be generated after injection of mouse iPSCs into Pax3 mutant mouse blastocysts, creating a niche in which stem cells can occupy and form skeletal muscle in the limb. This approach will provide evidence for the creation of entire skeletal muscle by mouse iPSCs in vivo. In addition, (2) we will generate a humanized skeletal muscle using Pax3 mutant mouse embryos via in utero injection of human iPSCs in combination with Pax3 mutant embryos and iPSCs. This concerted approach will help us to create iPSC-derived skeletal muscle and myogenic cells in vivo that can be transplanted into patients for a definitive cure of myopathic diseases and muscle injuries. In addition, the humanized skeletal muscle in mice will serve as an animal model to study the characteristics and regeneration characteristics of the human skeletal muscle diseases and responses to pharmacological agents and to provide a proof of concept for generating patient-derived cell and tissue sources for autologous muscle transplantation. .
Lay summary The innovation in our approach is to create humanized skeletal muscle and myogenic stem cells in animals with high efficiency. The creation of humanized skeletal muscle, healthy copies of a patient?s own skeletal muscle and myogenic stem cells, would revolutionize the field of regenerative medicine and transplantation for patients suffering myopathic diseases and muscle injuries.
